br Acknowledgements br Introduction Hepatocellular carcinoma

Acknowledgements
Introduction Hepatocellular carcinoma (HCC), the second leading cause of cancer deaths worldwide (Torre et al., 2015), arises in people with chronic liver disorders and inflammation. Most patients are not suitable for hepatic resection or transplantation. To treat these patients, chemotherapeutic agents, such as cisplatin, oxaliplatin, gemcitabine, and 5-fluorouracil, have been used, but they have low response rates. Sorafenib is the only agent approved for treating advanced HCC, but it has a limited effect on clinical outcome (Chen et al., 2015). Therefore, it is urgent to develop new approaches to treat HCC. Immunotherapy has been regarded as the best hope for cancer therapy, but the clinical benefits vary widely (Prieto et al., 2015; Koyama et al., 2016). Immune checkpoints, such as CTLA-4, PD-1 and TIM-3, have been the most widely studied targets for immunotherapy. For application to HCC, a recent phase I/II trial of a PD-1 antibody, CT-011, was halted because of slow accrual (ClinicalTrials.gov, Identifier: NCT00966251). A more appropriate immunotherapy for HCC needs to be found (Prieto et al., 2015; Chen et al., 2015). Infiltration of various immune cholesterol absorption inhibitor leads to an immunosuppressive microenvironment that is responsible for HCC initiation and progression; macrophages are a primary component of these cells (Sun and Karin, 2012; Li et al., 2017; Baeck et al., 2012). CCR2 is a feature of inflammatory monocytes, which are circulating precursors of tissue macrophages. In acute or chronic inflammation, CCR2 controls trafficking of bone marrow monocytes into the bloodstream and their migration to inflammatory sites (Kurihara et al., 1997). The CCL2/CCR2 axis and corresponding macrophage infiltration are involved in liver pathology, including acute and chronic hepatitis, cirrhosis, tumor progression, and metastasis (Huang et al., 2015; Li et al., 2017; Marra and Tacke, 2014), making it a potential immunotherapeutic target for liver cancer. CCR2 antagonists have been developed and evaluated in pre-clinical and clinical trials mainly due to the need for eliminating monocytes/macrophages, which would ameliorate inflammation associated with diseases. Although various agents have been discovered, and several have entered clinical trials, none have proved effective due to the selectivity of other chemokine receptors, their lack of potency in binding to rodent receptors, and poor PK/PD profiles (Struthers and Pasternak, 2010; Zimmermann et al., 2014). In the present study, we identified a natural product, 747, from Abies georgei (Fig. 1A), as a CCR2 antagonist and, with murine HCC models, evaluated it for activity in blocking CCL2/CCR2-mediated recruitment of monocytes/macrophages and for therapy of liver cancer. Further, we determined the effect of 747 combined with low-dose sorafenib for treatment of HCC in mice.
2. Materials and Methods
Results
Discussion Due to its involvement in various biological and pathological functions, CCR2 is an attractive target for drug discovery. However, development of CCR2 antagonists has been disappointing because of receptor redundancy, species differences, selectivity of other chemokine receptors, and limited clinical efficacy (Zimmermann et al., 2014). Since none of the present CCR2 antagonists has passed clinical trials, new concepts and strategies are needed. Natural products and their pharmacophores have been considered in the drug development process, and, from 1981 to 2014, they have accounted for nearly half of all newly approved drugs (Newman and Cragg, 2016). In the present study, a CCR2 antagonist, 747, was selected from a natural product library. 747 binds to human and mouse CCR2 with high affinity and selectivity relative to other chemokine receptors, particularly CCR5, which shares 72% homology (Zhao, 2010). Further, 747 binds to the major pocket of CCR2 through extracellular loops, whereas, among the mutants, only N199A mutant receptors decrease the binding affinity of 747 with CCR2. As determined from docking results, 747 forms hydrogen bonds with residues H121, F194, N199, N266, and T267, which partially explains why a single mutation may not show an appreciable effect on binding. Thus, 747, a CCR2 antagonist with a structure different from current antagonists, has antitumor potential and provides new insight for discovery of CCR2 antagonists.